A transmission electron microscope (TEM) transmits an electron beam through a thin sample and detects changes in the electron beam caused by interactions between the electrons and the sample. The electrons are typically accelerated to energies of between 80 and 300 keV. The acceleration is preferably done by maintaining the electron source at a high voltage and accelerating the electrons toward a sample at ground potential. Deflectors, lenses, and aberration correctors may manipulate the beam to irradiate the sample. The sample is sufficiently thin to be partially transparent to the electrons.
Because the transparency to electrons depends on the atomic number of the element, the typical sample thickness will vary with the nature of the sample. For example, samples composed of metals or semiconductors, which include many atoms having high atomic numbers, may be about 30 nm thick. Sample of biological materials are typically thicker, for example, about 200 nm thick. Even thicker samples, for example, 1 μm samples may be used in some applications.
Some electrons pass through the sample unhindered; some electrons are diffracted; some electrons lose energy; and some electrons are absorbed. Different techniques utilize different characteristics of the transmitted electrons to determine different information about the sample. A TEM can produce images that show structural features as in the nanometer or even sub-nanometer range and can also provide information about the composition of the sample.
After the electrons pass through the sample, they are detected by photographic film or by an electron detector that produces an electronic signal representative of the number and/or energy of electrons that impinge on the detector. Most TEM electron detectors detect the electrons indirectly: the electrons impinge on a phosphor, which emits light that is detected by the detector. Indirect detection prevents damage to the semiconductor detector by the impact of the high-energy electrons. The light impinges on a semiconductor detector and creates electron-hole pairs. In many indirect semiconductor detectors, the electrons accumulate in an array of capacitors that form part of a charged-coupled device (CCD). The accumulated charge on each capacitor is related to the number of electrons that impinged on the corresponding pixel. To read the charge on each pixel to obtain a complete image, the charge accumulated at each pixel is moved to the next pixel, like a bucket brigade, and eventually out of the detector where it is digitized and stored in a computer memory. The extra step of converting the electrons to light reduces the detector resolution because the light from the phosphor spreads before it reaches the CCD detector. Another disadvantage of the CCD detector is the time required to read out the array of pixels, and the limited field of view provided.
Some semiconductor detectors directly detect electrons, using active or passive pixel sensors, instead of a CCD. For example, a monolithic active pixel sensor (MAPS) implemented using complementary metal oxide silicon (CMOS) technology provides for a high-resolution detector with fast read out. In an active pixel sensor, each pixel can incorporate many of the functions required for particle detection, i.e., charge generation and collection, pre-amplification, pulse shaping, analog-to-digital conversion, noise discrimination and signal integration.
Active pixel sensors are described, for example, by R. Turchetta et al. in U.S. Pat. Pub. No. 2006/278943, for “Accelerated particle and high energy radiation sensor,” which is hereby incorporated by reference. Another type of active pixel sensor, is described in A. R. Faruqi et al., “Evaluation of a Hybrid Pixel Detector for Electron Microscopy.” Ultramicroscopy, vol. 94, 2002, pp. 263-276. In the hybrid active pixel detector of Faruqi et al., the sensitive read-out circuitry is located below the portion impinged by the electrons and is therefore protected from the electron impact. In another type of direct electron detector, a double-sided strip detector (DSSD), the read-out system is not in line with the electron beam and so does not degrade. The DSSD uses collection strips on the top and bottom of the detector and can provide information about each electron event as it occurs, allowing the processor to determine, for example, when two electrons strike at about the same time.
The sample in a TEM is maintained in a vacuum, because air or other gas molecules in the sample chamber would scatter the electrons in the beam. Biological samples, which contain a large amount of water would quickly degrade in a high vacuum environment. Some techniques for preserving biological samples for observation in a TEM include staining or other fixing techniques that can introduce artifacts into the observation. Artifacts are observed features that are a result of the imaging process, and are not naturally occurring in the sample. A preferred preservation technique, which better maintains the integrity of a biological sample, is to rapidly freeze in a vitrification process that produces amorphous ice, and to observe it at cryogenic temperature. Cryogenic transmission electron microscopy (Cryo-TEM) entails the observation of samples at cryogenic temperatures, typically liquid nitrogen or liquid helium temperatures, on a transmission electron microscope. Cryo-TEM allows viewing a specimen in its native state without introducing artifacts into the observation during a fixing process.
Images from a cryo-TEM, however, are degraded by various processes that occur during the observation. For example, the impact of high energy electrons in the beam heats the thin sample which, and after a short period of observation can cause bubbles in the sample. Another problem with cryo-TEM is that samples accumulate a static electric charge at the beginning of imaging, which produces significant streaking in the corners of the image. The charge dissipates after a brief period. To avoid the charging artifact, a user will sometimes pre-illuminate a sample to eliminate charging before forming an image. Users also use spot-scan imaging to overcome charging and beam induced movement. Both these methods have the undesirable effect of reducing the time that the electron beam is producing useful information, since the pre-illumination begins to heat the sample, but does not produce useful image data.